The invention relates to the production of hybrid seed in wheat by using a deacetylase gene to obtain conditional sterility. Vectors are described comprising a deacetylase gene under the control of promoters directing expression selectively in stamen cells in wheat, which are particularly suited for the production of wheat plants which can be made male-sterile upon application of N-acetyl-PPT.
All documents cited herein are included herein by reference.
Deacetylases are capable of deacetylating acetylated toxins, such as N-acetyl-phosphinothricin (N-Ac-PTC or N-Ac-PPT), intracellularly, whereupon the cytotoxic activity of the toxins is restored (formation of PTC or PTC). U.S. Pat. No. 5,650,310, U.S. Pat. No. 5,668,297, U.S. Pat. No. 5,767,370, and U.S. Pat. No. 5,767,371, describe deacetylase genes, for the production of phosphinothricin or phosphinothricyl-alanyl-alanine, as well as processes for their isolation and use. Furthermore deacetylase genes are disclosed therein which are isolated from Streptomyces viridochromogenes (dea) and from E. coli (ArgE). The use of such genes for the production of plants with selectively destroyable plant parts is described therein. More specifically, a method is disclosed for the production of conditionally male-sterile plants. This is achieved by introducing a deacetylase gene, under the control of a tapetum-specific promoter into the genome of a plant. Upon application of N-Ac-PPT to the plant, in the tapetum cells, where the deacetylase is expressed, N-Ac-PPT is converted into PPT, which is toxic to the cells. The cytotoxic activity in the tapetum cells inhibits the development of microspores and renders the plant male-sterile.
WO 98/13504 describes the use of deacetylase genes for the production of female-sterile plants. WO 98/39462 describes a method of hybrid seed production using conditional female sterility, whereby a plant is made conditionally female-sterile by transformation with the ArgE gene linked to a female-preferential promoter.
WO 98/27201 describes novel genes encoding amino acid deacetylases isolated from Stenotrophomonas sp. (deac) and from Comamonas acidovorans (ama) and their use in the production of transgenic plants.
The high production cost of hybrid wheat has limited the use of commercial hybrids. The economic relationship between the additional costs of male sterilisation and seed production are up to now clearly unfavourable in breeding and agriculture. In contrast to crops such as maize, the hybrid seed production in wheat has been subject to considerable difficulties. The reproductive system of wheat, a self-fertilizer, is poorly adapted to cross-fertilization, which has led to poor hybrid seed set and consequently low yields. This has resulted in a high price for the hybrid seed on farm-level. In comparison to line varieties, the yield advantage of hybrids in wheat is up to now not sufficient enough to compensate high seed cost. (Picket, 1993, In: Hybrid Wheat-Results and Problems, Avances in Plant Breeding, Paul Parey Scientific Publishers, Berlin and Hamburg).
In the last few years, mainly due to an improvement in agricultural production steps, interest in hybrid wheat has resurfaced, and first hybrid wheat varieties have been marketed.
Traditional large-scale hybrid seed production is accomplished by planting separate rows or blocks of female parent lines and male parent lines as pollinators. Only the seed produced on the female parent rows or blocks is harvested. To ensure that this seed is hybrid seed, uncontaminated with selfed seed, pollination control must be implemented. Known pollination control methods are generally chemical, using a hybridising agent, or genetic. Genetic methods have been developed using either natural or molecular genes, based on either cytoplasmic or nuclear sterility factors. Neither the cytoplasmic male-sterility nor the chemical male-sterility are ideal and both show important economical, practical and/or enviromental drawbacks. The successful use of cytoplasmic male-sterility for commercial hybrid seed production requires a stable male cytoplasm and the availability of reliable restorer genes which is a limiting factor in the wheat system presently used based on the T. timopheevi system (Wilson, J. A. and Ross, W. M. (1962). Wheat Information Service, Kyoto University 14: 29-30; Wilson, J. A. and Ross, W. M. (1962b). Crop Science 2: 415-417). Furthermore such a system of cytoplasmic male sterility requires three lines to produce a single crossed hybrid: the A line (male-sterile), the B line (male-fertile maintainer), and an R line (male-fertile with restorer genes). Three-way crosses produced with cytoplasmic-genetic male sterility involve maintenance and production of four lines, the A and B lines of one inbred and male-fertile inbreds of the other two.
Alternatively, the production of hybrid seed can be achieved using chemicals that block or kill viable pollen formation. These chemicals, called gametocides, are used to impart a transitory male-sterility. The expense and the availability of the chemical, the reliability of the applications under different environments and the toxicity of the chemical to the environment however, limit its use in the production of hybrid seed.
Molecular methods for hybrid seed production offer a new promising alternative. Male-sterile wheat plants were obtained by introducing the barnase gene under the control of tapetum-specific promoters from corn and rice (De Block et al., 1997, Theor. Appl. Genet. 95:125-131). Such transformed plants are functionally male-sterile and can be used for the production of hybrid seed by crossing with male-fertile pollinator plants, containing corresponding restorer genes (to ensure restoration of fertility in the hybrid; as described in EP 344029, U.S. Pat. No. 5,689,041, U.S. Pat. No. 5,792,929, U.S. Pat. No. 5,723,763). The genetically engineered male sterility, whether it works through anti-sense or RNAse, can only be maintained in a heterozygous state. The heterozygous female parent, of which only 50% will be male sterile, must be planted in rows next to the pollen donor (male) parent and the other 50% of fertile female parent have to be removed. By genetic engineering it is possible to link the male sterility gene to a herbicide resistance gene and remove the fertile plants by spraying the corresponding herbicide. Still this system has economical drawbacks. Due to the fact that only 50% of the female plants can be used for hybrid seed production, the female parent rows must be planted at double density in order to obtain the same yield per acre. This will cause yield loss because of competition between plants. The herbicide spray also induces yield loss as resistant plants are never 100% resistant to the herbicide. The considerable costs for the chemical and for the xe2x80x9crestorerxe2x80x9d breeding program makes that this hybrid seed production system is economically not ideal for wheat. To achieve a more economical hybrid seed production system for wheat and potentially other cereal crops, the use of a conditional male-sterility system, whereby plants can be maintained in their male fertile form and no restorer genes are needed would be desirable. It remains to be described how such a conditional male-sterility system can optimally be developed for wheat and other cereal crops like barley, rye and oats.
The invention relates to a method for producing conditionally male-sterile wheat plants, which method comprises 1) introducing into the genome of a wheat cell or tissue a foreign DNA comprising a chimeric gene which comprises a DNA encoding a deacetylase, more specifically a deacetylase from Stenotrophomonas sp., under the control of a promoter directing expression selectively in stamen cells in wheat, and 2) regenerating a plant from the cell or tissue, which plant is conditionally male-sterile.
The invention further relates to a conditionally male-sterile wheat plant comprising, integrated into its genome, a foreign DNA comprising a chimeric gene which comprises:
a) a DNA encoding a deacetylase, more specifically a deacetylase from Stenotrophomonas sp., under the control of
b) a promoter directing expression selectively in stamen cells in wheat, such as the CA55 promoter, the T72 promoter or the E1 promoter;
whereby the wheat plant can be made male-sterile by application of an acetylated toxin, such as N-Acetyl-PPT, to the plant.
The invention further relates to a conditionally male-sterile wheat plant comprising, integrated into its genome, a foreign DNA comprising a chimeric gene which comprises a deacetylase coding sequence isolated from Stenotrophomonas sp., preferably from the organism deposited under accession No. DSM 9734 or a biologically active fragment or variant thereof; additionally or alternatively according to the present invention, the deacetylase coding sequence encodes the deacetylase corresponding to SEQ ID No. 8 described herein or a biologically active fragment or variant thereof; additionally or alternatively the DNA encoding a deacetylase of the invention comprises the sequence of SEQ ID No. 9, or a fragment or variant thereof and/or corresponds to a DNA sequence which hybridizes to SEQ ID No. 9 under standard stringent conditions.
The invention further relates to a method for producing composite hybrid seed in wheat, which comprises
producing seeds capable of growing into conditionally male-sterile plants, comprising, integrated into their genome, a foreign DNA comprising a chimeric gene which comprises a DNA encoding a deacetylase from Stenotrophomonas sp. under control of a promoter directing expression selectively in stamen cells in wheat,
interplanting these seeds with seeds capable of growing into male fertile plants,
inducing male sterility in the conditionally male-sterile plants by applying an acetylated toxin, such as N-acetyl PPT to the conditionally male-sterile plants; and
harvesting seed from both conditionally male-sterile plants and male fertile plants, which is composite hybrid seed.
The invention further relates to a method for producing pure hybrid seed in wheat by planting the above-described seeds capable of growing into conditionally male-sterile plants and seeds capable of growing into male-fertile plants in separate rows or blocks, and harvesting seed only from the pollinated conditionally male-sterile plants.
The invention further relates to a method for producing pure hybrid wheat seed by crossing conditionally male-sterile wheat plants, comprising integrated into their genome a DNA encoding a deacetylase from Stenotrophomonas sp. under control of a stamen selective promoter, with female sterile plants as described in U.S. Pat. No. 5,633,441 or U.S. Pat. No. 5,767,374 or conditionally female-sterile plants as described in WO 98/13504, which are male-fertile, and harvesting hybrid seed from the pollinated conditionally male-sterile plants.
The invention further relates to a conditionally male-sterile wheat plant comprising, integrated into its genome, a foreign DNA comprising chimeric gene which comprises a DNA encoding a deacetylase from Stenotrophomonas sp., under control of a stamen-selective promoter, and which further comprises a second chimeric gene which confers on the plant resistance to a herbicide.
The invention further relates to a conditionally male-sterile wheat plant, comprising the chimeric gene of the invention comprising a DNA encoding a deacetylase and further comprising a second chimeric gene which confers on the plant resistance to PPT. More specifically said second chimeric gene comprises a gene, such as the pat or bar gene, capable of converting PPT into N-acetyl-PPT. The N-acetyl-PPT so produced can be transported through the plant to the stamen cells in which the deacetylase is expressed, where it can be converted to PPT. It is understood that in this embodiment of the invention, specific destruction of the stamen cells can be induced by treatment of the plant with N-acetyl PPT or PPT.
The invention further relates to a method for producing pure hybrid seed in wheat, which comprises:
producing seeds capable of growing into conditionally male-sterile plants comprising, integrated in their genome, a foreign DNA comprising
a) a chimeric gene comprising a DNA encoding a deacetylase from Stenotrophomonas sp. under control of a promoter that directs stamen selective expression in wheat, and further comprising
b) a chimeric gene which confers on the plants resistance to a herbicide,
interplanting seeds capable of growing into conditionally male-sterile plants with seeds capable of growing into male fertile plants, that are not resistant to the herbicide,
inducing male-sterility in the conditionally male-sterile plants by applying an acetylated toxin, such as N-acetyl PPT, to the conditionally male-sterile plants;
allowing pollination of the male-sterile plants by the male fertile plants;
applying the herbicide to eliminate the male fertile plants; and
harvesting seed from the conditionally male-sterile plants.
The present invention also demonstrates that particular stamen selective promoters, particularly the CA55 promoter, the PE1 promoter, and the T72 promoter are particularly useful, when linked to a deacetylase gene, for the generation of conditionally male-sterile plants according to the invention.
The present invention further demonstrates that the deac system is particularly suited for obtaining inducible male sterility in cereal plants, such as barley, oats, rye and most particularly in wheat.